志贺菌和肠侵袭性大肠埃希菌LAMP检测方法的建立及应用

摘要

目的 建立针对侵袭性质粒抗原H(ipaH)编码基因的环介导等温扩增(LAMP)方法.方法 利用LAMP方法对4株志贺菌、1株肠侵袭性大肠埃希菌(EIEC)和20株其他肠道菌DNA在65℃恒温扩增30 min或60 min后观察结果.结果 4株志贺菌和EIEC可通过肉眼和电泳观察到阳性结果,而其他肠道菌为阴性.LAMP检测特异性与聚合酶链反应(PCR)相似,但敏感性比PCR高10倍.LAMP、PCR检出下限分别为1.2×10 2、1.2×103 CFU/mL.LAMP和分离培养法对70例食物中毒患者和健康者肛拭子标本检测结果符合率为100％.结论 LAMP由于具有快速和无需特殊仪器的优点,可广泛用于肛拭子标本志贺菌和EIEC的检测.%Objective To develop a loop-mediated isothermal amplification(LAMP) method targeting the invasion plasmid antigen H(ipaH) for the detection of Shigella and enteroinvasive Escherichia coli(EIEC).Methods DNA of four strains of Shigella, one strain of EIEC and twenty strains of other enteric bacterial were amplified under isothermal conditions(65℃) within 60 or 30 min,then the results were tested.Results Both naked eye and agarose gel electrophoresis could detect positive reaction in Shigella and EIEC,but not in other bacteria.The specificity of LAMP was similar to polymerase chain reaction(PCR) ,but the sensitivity of LAMP was 10 times higher than that of PCR.The detection limit of LAMP was 1.2 × 102 CFU/mL, whereas that of PCR was 1.2×103 CFU/mL.The coincidence rate of LAMP and isolated culture assay to detect 70 rectal swab samples from patients with diarrhea and healthy subjects was 100%.Conclusion Due to its rapidity and minimal equipment requirement, LAMP might be a useful and rapid detection method of Shigella and EIEC in rectal swab samples.